Antimicrobial peptides

ABSTRACT

Peptides which exhibit antimicrobial activity comparable to certain known antibiotics are provided. These peptides are related in sequence to amino acid sequences within Cathepsin G. A broad spectrum bactericidal peptide disclosed herein is RPGTLCTVAGWGRVSMRRGT (SEQ ID NO:22). It is active against Pseudomonas aeruginosa, Neisseria gonorrhoeae and Staphylococcus aureus. RRENTQQHITARRAIRHPQY (SEQ ID NO:19) and GKSSGVPPEVFTRFVSSFLPWIRTTMR (SEQ ID NO:20) also exhibited potent activity against P, aeruginosa strains, including clinical isolates. IIGGR (SEQ ID NO:1) and IVGGR (SEQ ID NO:2) act against both gram-negative and gram-positive bacterial strains. HPQYNQR (SEQ ID NO:3) and certain related peptides are also active against both gram-negative and gram-positive bacteria, including, but not limited to, strains of Escherichia coli, Neisseria gonorrhoeae, Staphylococcus aureus, Capnocytophage sputigena and Pseudomonas aeruginosa. The peptides of the present invention will be useful in pharmaceutical compositions useful in the treatment of prophylaxis of infections.

GOVERNMENT RIGHTS

This invention was made, in part, with funding from the NationalInstitutes of Health and from the Department of Veterans AffairsResearch Service. The United States Government may have certain rightsin this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 07/541,635,filed Jun. 21, 1990, now abandoned, which is incorporated by referenceherein.

TECHNICAL FIELD

The field of this invention is the area of antimicrobial peptides withactivity against a broad range of Gram-negative and Gram-positivebacteria and fungi. The antimicrobial peptides of this invention areuseful for inhibiting microbial growth and in pharmaceuticalcompositions for treatment or prevention of infections and for thetreatment and/or prevention of gingivitis.

BACKGROUND OF THE INVENTION

Microbes which invade the human body are challenged by several defensemechanisms. The nature of the defense mechanisms which any given microbefaces depends on the genetic makeup and the physiologic state of thehost as well as the portal of entry of the invading microorganism.

Host defenses include mechanical factors and chemical factors.Mechanical factors which help protect epithelial surfaces include thewashing action of bodily fluids, including tears, saliva and urine,trapping on mucous layers, removal by cilia and elimination by coughing,sneezing or desquamation. A further mechanical defense is offered by thephysical integrity of the skin, although mucous membranes can bepenetrated by some pathogens.

Chemical defense factors include the acidity of gastric secretions,unsaturated fatty acids on the skin which kill certain bacterialspecies, lysozyme in tears, saliva and nasal secretions, iron-bindingproteins at the mucosal surface, transferrin in serum, and spermine insemen. Secretions of the mucous membranes also contain antibodies,especially those of the IgA class. Microbial antagonism betweendifferent potentially pathogenic bacteria, fungi and yeast strainsoccurs at the level of competition for nutrients and through theproduction of inhibitory substances; this antagonism affords furtherprotection to the host.

If the barriers of the skin or mucous membranes are crossed,immunological factors (e.g., antibodies) which are specific to themicroorganism as well as nonspecific cellular defenses come into play.In addition, there are some chemical factors which also play a role inhost defense, especially transferrin, which chelates available iron onwhich microorganisms are dependent.

Nonspecific cellular defenses in the form of phagocytic white bloodcells from local tissues and the bloodstream respond to an invadingmicrobe. Polymorphonuclear leukocytes (PMNs) actively phagocytizeparticulates such as bacterial or fungal cells. PMNs are the first classof phagocytic cells recruited to the site of infection or inflammation.The PMNs contain azurophilic or primary granules, which containlysosomal proteases, myeloperoxidase, lysozyme and certain antimicrobialproteins. Secondary granules within these cells contain alkalinephosphatase, lactoferrin and lysozyme. Stores of glycogen within thePMNs provides for energy through glycolysis so that the cell canfunction in an anaerobic environment.

Adherence of a particle to the surface of a phagocytic cell initiatesphagocytosis; the particle enters the cytoplasm in a phagocytic vacuole.This triggers a respiratory burst and the generation of microbicidalmetabolites; the primary granule fuses with the phagocytic vacuole toform a digestive vacuole called the phagolysosome. Intracellular killingof the ingested microorganism occurs as a result of oxygen-dependent andoxygen-independent mechanisms. The oxygen-dependent bactericidalhalogenating system uses granule myeloperoxidase, hydrogen peroxide andchloride ion to kill bacteria and viruses via either halogenation ofcellular or viral constituents or via reactive oxygen intermediates.Oxygen-dependent killing can also proceed by direct reduction ofmolecular oxygen via the cytochrome b-oxidase system (reviewed by Orkin(1989) Ann. Rev. Immunol. 7:277-308). Oxygen-dependent killingmechanisms are reviewed by Beaman and Beaman (1984) Ann. Rev. Microbiol.38:27-48.

The primary granules contain three major groups of antibacterialproteins. The first group includes catalytically active proteins whichare only weakly antibacterial when tested individually in purified form.Examples from this group include lysozyme, elastase and collagenase.These enzymes probably participate in the digestion of microorganismskilled by other mechanisms, but elastase, for example, is believed topotentiate killing by the halogenating system. The second category ofgranule proteins includes those with catalytic activity and bactericidalactivity which is independent of the catalytic activity. An example isthe chymotrypsin-like neutral protease of human neutrophils. A thirdgroup contains bactericidal members which lack known catalytic activity;such a protein class has been purified from rabbit neutrophils. Includedin this class are defensins and cationic antibacterial proteins.

Some cationic antibacterial proteins are of relatively high molecularweight (greater than about 25 kDa) and kill certain Gram negativebacteria such as Escherichia coli, Salmonella typhimurium andPseudomonas aeruginosa by damaging the cytoplasmic membrane, leading toincreased membrane permeability. Human bactericidal/permeabilityincreasing protein (BPI) is a strongly basic protein with molecularweight of about 59 kDa. It is believed that when bound to the outermembrane of susceptible bacterial cells, hydrophobic channels throughthe outer envelope are exposed, and as a secondary effect, there is aselective activation of autolytic enzymes including phospholipase andpeptidoglycan hydrolases. Gram positive bacteria, certain Gram negativebacteria and fungi are not affected by BPI in vitro.

Low molecular weight cationic proteins (10 kDa to 25 kDa) have beenreported which inhibit the multiplication of such Gram positive bacteriaas Staphylococcus aureus (Root and Cohen (1981) Rev. Infect. Dis.3:565-598). In addition, cationic proteins with fungicidal activity havebeen identified in alveolar macrophages. It is believed that cationicproteins are most efficient in killing phagocytized microorganisms incombination with other microbicidal defense mechanisms (Elsbach andWeiss (1983) supra).

Generally defensins are relatively small polypeptides of about 3-4 kDa,rich in cysteine and arginine. Gabay et al. (1989) Proc. Natl. Acad.Sci. USA 86:5610-5614, used reverse phase HPLC to purify 12 majorpolypeptides from the azurophil granules of human PMNs; purifiedproteins were analyzed individually for antimicrobial activity and forN-terminal amino acid sequence. A 4 kDa defensin (HNP-4) and a 29 kDapolypeptide named azurocidin were purified and shown to possess broadspectrum antimicrobial activity. Defensins as a class have activityagainst some bacteria, fungi and viruses. They are also reported to havecytotoxic activity against transformed cells. Selsted et al. (1985) J.Clin. Invest. 76:1436-1439, presents a sequence comparison of human andrabbit defensins. The defensins are believed to have molecularconformations stabilized by cystine infrastructure, which are essentialfor biological activity.

Granzymes are a family of serine proteases in the granules of cytolyticlymphocytes. Proteolytic enzymes are believed to function incell-mediated cytoxicity; some of the genes have been cloned, andsequence information is available. Within the granzyme family there isat least 38% amino acid sequence identity. Human lymphocyte protease has73% amino acid sequence identity to mouse granzyme B (Jenne and Tschopp(1988) Immunol. Reviews 103:53-71).

Cathepsin G (Cat G) is a granule protein with chymotrypsin-likeactivity; it is also known as chymotrypsin-like cationic protein. Cat G(Odeberg and Olsson (1975) J. Clin. Invest. 56:1118-1124) and threeother mutually homologous polypeptides called defensins are activeagainst a broad spectrum of gram positive bacteria, gram negativebacteria and fungi (Shafer et al. (1986) Infect. Immun. 54:184-188;Shafer et al. (1988) Infect. Immun. 56:51-53; Drazin and Lehrer (1977)Infect. Immun. 17:382-388; Ganz et al. (1986) Semin. Respir. Infect.1:107-117). Sensitive bacteria include Capnocytophaga sputigena,Escherichia coli, Listeria monocytogenes, Neisseria gonorrhoeae,Pseudomonas aeruginosa and S. aureus. All of these pathogens, with thenotable exceptions of P. aeruginosa and C. sputigena, are only sensitiveto both enzymatically-active and -inactive cathepsin G (Miyasaki andBodeau (1991) J. Clin. Invest 87:1585-1593; Wasiluk et al. (1991)Infect. Immun. 59:4193-4200 and Table 11 herein). P. aeruginosa and C.sputigena are only sensitive to enzymatically-active cathepsin G. It isnot clear, however, if cathepsin G-killing of these two pathogensrequires degradation of bacterial proteins or whether an intact activesite is needed to align antibacterial domains of cathepsin G with thebacterial target.

Gabay et al. (1989) supra, has reported antibacterial activities of anumber of proteins isolated from human PMNs, including cathepsin G andelastase, and has given the amino terminal sequence of these and otherproteins. The N-terminal five amino acids of elastase and Cat G areidentical; further sequences have significant relatedness. Cat G alsoexhibits significant sequence similarity to chymotrypsin, which is notknown to exhibit antimicrobial activity similar to that of Cat G.

The sequence of human Cat G is known, and the gene has been cloned fromhuman leukemic cell line U937 (Salvesen et al. (1987) Biochemistry26:2289-2293). Sequence analysis of the cDNA revealed significantsequence identity to rat mast cell proteinase (47%) and to an activatedmouse cytotoxic lymphocyte product (56%).

Another class of antimicrobial polypeptides are those known asmagainins; at least five proteins can be isolated from the skin of theAfrican clawed frog (Xenopus laevis). The natural proteins are activeagainst a broad range of microorganisms including bacteria, fungi andprotozoans (Zasloff (1987) Proc. Natl. Acad. Sci. USA 84:5449-5453). Thebroad spectrum antimicrobial activity is present in synthetic peptidesand in certain truncated analogs of the natural proteins. Derivatives ofabout 19 to about 23 amino acids have antibacterial activity as measuredusing Escherichia coli. In the protozoan Paramecium caudatum treatedwith the magainin peptides, there is disruption of membrane functions.The configurations of the bioactive peptides can be modeled asamphiphilic alpha-helices and are sufficiently long to span a lipidbilayer. (Zasloff et al. (1988) Proc. Natl. Acad. Sci. USA 85:910-913).Spanning a lipid bilayer is believed to require at least 20 amino acidresidues in an alpha-helical configuration (Kaiser and Kennedy (1987)Ann. Rev. Biophys. Chem. 16:562-581). The sequence of a representativemagainin peptide is GIGKFLHSAKKFKAFVGEIMN (SEQ ID NO:48) (Zasloff et al.(1988) supra).

SUMMARY OF THE INVENTION

It is an object of this invention to provide peptides with antimicrobialactivity. The antimicrobial peptides of the present invention containfrom about five to about twenty-six amino acids joined in a linear arrayby peptide bonds.

An object of the present invention is a broad spectrum bactericidalpeptide, termed CG 117-136 herein, which has the sequenceRPGTLCTVAGWGRVSMRRGT (SEQ. ID NO:22). Further objects are additionalbactericidal peptides, particularly effective for P. aeruginosa but notfor S. aureus or N. gonorrhoeae, which peptides are termed CG 61-80 andCG 198-223, herein. CG 61-80 has the sequence RRENTQQHITARRAIRHPQY (SEQID NO:19) and CG 196-223 has the sequence GKSSGVPPEVFTRVSSFLPWIRTTMR(SEQ ID NO:26).

In other embodiments, the peptides comprise the amino acid sequencesIIGGR (SEQ ID NO:1), IVGGR (SEQ ID NO:2), IIGGRESRPHSRPYMAYLQI (SEQ IDNO:16) and HPQYNQR (SEQ ID NO:3). The peptide of the sequenceIIGGRESRPHSRPYMAYLQI is particularly preferred. A consensus sequence forpeptides related to HPQYNQR (SEQ ID NO:3) has been formulated: HX₁ X₂ X₃X₄ X₅ X₆, where X₁ is proline, histidine or alanine; X₂ is asparticacid, asparagine, glutamic acid, glutamine, alanine, serine, threonine,isoleucine, valine, histidine, tyrosine, arginine, methionine oxide ormethionine sulfone; X₃ is tyrosine, phenylalanine, tryptophan orbeta-naphthyl-alanine; X₄ is asparagine or alanine; X₅ is glutamine,proline, N-methyl alanine, or alanine; and X₆ is arginine, lysine,alanine or NH₂ or OH (SEQ ID NO:4).

A peptide fitting the consensus sequence preferably has at position 3(as X₂ above) a nonbulky, hydrophilic amino acid capable of hydrogenbonding, glutamine or proline at position 6 (as X₅ above) and lysine orarginine at position 7 (as X₆ above). Preferably amino acid 3 isglutamine, alanine, glutamate, asparagine, or aspartate. Antimicrobialpeptide sequences whose sequences fall within the consensus sequenceinclude, but are not limited to, HPQYNQR (SEQ ID NO:3), HPAYNPK (SEQ IDNO:5), HPAYNPR (SEQ ID NO:6) and HPAYNQR (SEQ ID NO:7). Additionalantimicrobial peptide sequences related to HPQYNQR include, but are notlimited to, HPQYAQR (SEQ ID NO:8), HPQYNQA (SEQ ID NO:9), HPQYNAR (SEQID NO:10), HPAYNPR (SEQ ID NO:6), HAQYNQR (SEQ ID NO:11), and HPQYNQ(SEQ ID NO:12) and RHPQYNQR (SEQ ID NO:13). These peptides possessmicrobicidal activity for Gram positive and Gram negative bacteria. Theoligopeptide corresponding in sequence to amino acid 77-96 of maturecathepsin G, HPQYNQRTIQNDIMLLQLSR (SEQ ID NO:14), is not significantlybactericidal, however, for P. aeruginosa, N. gonorrhoeae or S. aureus.

An object of the present invention is to provide antimicrobial peptideswhich are useful as bactericides and/or bacteriostats, useful, forexample, for inhibiting microbial growth in a variety of solutions andsterile solutions, such as contact lens solutions, herbicidal solutions,hazardous or refuse waste streams, surface disinfectant solutions andoil recovery fluids.

A further object of the invention is to provide therapeuticcompositions, suitable for human, veterinary, agricultural orpharmaceutical use, comprising one or more of the antimicrobial peptidesof the present invention and a suitable pharmacological carrier. Suchtherapeutic compositions can be formulated as understood in the art,e.g., for topical or aerosol application, for controlling and/orpreventing infection by Gram positive or Gram negative bacteria orfungi. Preferably, the antimicrobial peptides of the present inventionare used in the treatment of infections by Gram-negative orGram-positive bacteria. The antimicrobial peptides of the presentinvention, when used in therapeutic compositions, will not havesignificant immunogenic activity. In vitro antimicrobial activity of theoligopeptides of the present invention is an accurate predictor of invivo antimicrobial activity.

Pharmaceutical compositions contain a therapeutically effective amountof an antimicrobial peptide. A therapeutically effective amount of anantimicrobial peptide can be readily determined according to methodsknown in the art. Pharmaceutical compositions are formulated to containthe therapeutically effective amount of an antimicrobial peptide and apharmaceutically acceptable carrier appropriate for the route ofadministration (topical, gingival, intravenous, aerosol, localinjection) as known to the art. For agricultural use, the compositioncomprises a therapeutically effective amount of an antimicrobial peptideand an agriculturally acceptable carrier suitable for the organism(e.g., plant) to be treated. Preferably for use in a pharmaceuticalcomposition, the antimicrobial peptide will have an ED₅₀ in vitro lessthan about 10⁻³ M. The skilled artisan can readily determine atherapeutically effective amount against a target bacterial strain, forexample, based on the ED₅₀ using the methods disclosed herein and theteachings of the art.

Therapeutic compositions may be administered by topical, dental rinse,aerosol or intravenous application, or by local injection for thecontrol or prevention of infection or control of tumor cell growth, byany means known to the art.

The IIGGR-related antimicrobial oligopeptides, including IIGGR (SEQ IDNO:1), IVGGR (SEQ ID NO:2) and IIGGRESRPHSRPYMAYLQI (SEQ ID NO:16), ofthe present invention may also be used to kill or control the growth oftumor cells or virus-infected cells. In such applications, thesepeptides will be particularly useful when coupled to antibodies or othermolecules which are specific for the target tumor cell or virus-infectedcell so that the peptide acts specifically on the tumor orvirus-infected cell.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the bactericidal activity of clostripain digests ofhuman neutrophil cathepsin G and elastase. Proteinases were digested asdescribed in the Examples, lyophilized, dissolved in HBSS (pH 7.5), andtested (50 μg/ml) for antibacterial activity against S. aureus strain8325-4. Each data point represents the mean of three samples with <5%variance between each.

FIG. 2 shows Reverse Phase-HPLC fractionation of cathepsin G peptidesobtained from clostripain fragmentation. Low M_(r) peptides obtained bygel filtration chromatography were lyophilized, dissolved in 250microliters of 0.1% (v/v) TFA and subjected to RP-HPLC as described inthe Examples. Peptides were eluted in several peaks (1-10). These werefurther purified, separately, by RP-HPLC as described herein. Onlypeptides in peaks 6 and 7 were found to exert antibacterial action invitro against S. aureus and N. gonorrhoeae. FIG. 2(A), cathepsin Gdigest fractionation; FIG. 2(B), rechromatography of peak 6; FIG. 2(C),rechromatography of peak 7.

FIG. 3 illustrates the bactericidal activity of synthetic peptidesderived from cathepsin G. Synthetic peptides (0-100 μg) were testedagainst N. gonorrhoeae and S. aureus as described in the Examples. Eachdata point represents the mean of three determinations from threeseparate experiments using the same lot of synthetic peptide. FIG. 3(A),peptides IIGGR (SEQ ID NO:1) vs. IVGGR (SEQ ID NO:2); FIG. 3(B),peptides HPQYNQR (SEQ ID NO:3) vs. APQYNQR (SEQ ID NO:15).

FIG. 4 illustrates the pH dependency for the bactericidal activity ofHPQYNQR (SEQ ID NO:3). Aliquots of peptide (100 μg) were assayed forantibacterial activity in incubation with either of the two testbacteria at different pH values, as described herein. Each data point isthe average of two determinations from three separate experiments.

FIG. 5 compares the bactericidal capacities of Cat G synthetic peptidesand certain antibiotics. N. gonorrhoeae strain FA 102 was exposed to5.0×10⁻⁴ M of IIGGR (SEQ ID NO:1), HPQYNQR (SEQ ID NO:3), penicillin G,chloramphenicol, tetracycline, streptomycin, kanamycin, and a mixture ofthree partially purified human defensins in HBSS (pH 7.5). After 30 minat 37° C. the samples were plated onto GCB agar, incubated for 48 hr,and colonies counted. There were no gonococcal colonies after 48 hr ofincubation.

FIG. 6 illustrates the bactericidal action of Cat G synthetic peptides(HPQYNQR, Ac-HPQYNQR (SEQ ID NO:3)) (see Table 2), and human granzymeB-derived peptide (HPAYNPK (SEQ ID NO:5)) under conditions of differentpH, FIG. 6A and ionic strength, FIG. 6B, and at different time pointsFIG. 6C. The synthetic peptides (5×10⁻⁴ M) and purified, enzymaticallyinactive Cat G (1.8×10⁻⁶ M), were tested against S. aureus in HBSSmodified to different pH FIG. 6A and ionic strengths FIG. 6B. Standardionic strength HBSS was used in FIG. 6A, while pH 7.5 HBSS was used inFIG. 6B. The peptides and Cat G preparations are identified in theinserts. The results are mean values from three separate experiments.FIG. 6C Time course of killing S. aureus by Cat G and granzyme B-derivedsynthetic peptides. Synthetic peptides (5×10⁻⁴ M) were tested forbactericidal action over a 120 min period in optimal HBSS. The resultsare from three separate experiments and mean values.

FIG. 7 graphically illustrates the in vitro bactericidal activity ofvarious Cat G-related oligopeptides against C. sputigena ATCC 33123. 10⁸cells/ml were incubated 2 h in 1/100 strength HBSS at a concentration of100 μg/ml for cathepsin G, or at 500 μg/ml for peptides derived fromcathepsin G. The y-axis gives bactericidal activity, as measured by logs(base 10) of lost viability.

FIG. 8 illustrates the complete amino acid sequence (SEQ ID NO:41) forthe mature human cathepsin G, as deduced from analysis of its cDNA. Inthe chymotrypsin nomenclature, it displays the charge relay profile ofHis57 Asp102 Ser195 that is typical of serine proteases; the chargerelay system amino acids are identified with an asterisk (*). The Ser195residue (residue 181 in the mature cathepsin G protein) is the target ofphosphorylation by DFP, resulting in irreversible inhibition ofchymotryptic activity. The residues lining the primary specificitypocket of cathepsin G are marked with #.

FIG. 9 presents hydrophobicity analysis for peptide CG 117-136 (SEQ IDNO:22) and the corresponding hydrophobicity-hydrophilicity plot for theamino acid sequence.

FIG. 10 presents a comparison of the amino acid sequence of the broadspectrum antibacterial peptide CG 117-136 (SEQ ID NO:22) with relatedsequences in other antibacterial proteins and serine proteases. The dotsrepresent amino acid residue identity with the CG 117-136 sequence. Thepartial sequences of human granzyme B, mouse granzyme B, CAP 37, humanelastase, proteinase 3 and bovine chymotrypsin correspond to SEQ ID NOs:42, 44, 45, 46 and 47, respectively.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, an oligopeptide is composed of from about five to abouttwenty-seven amino acids linked together by peptide bonds in a lineararray. The peptide may be in a linear conformation or it may assumesecondary structure. A cyclic peptide derivative can also haveantimicrobial activity, and thus is a functional equivalent of theantimicrobial peptides of the present invention. Sequences areconventionally given from the amino terminus to the carboxyl terminus.The peptides of the present invention have antimicrobial activity bythemselves or when coupled to another molecule, e.g., polyethyleneglycol or a carrier protein such as bovine serum albumin, so long as thepeptides are positioned such that they can come into effective contactwith the target cell.

Table 1 presents most abbreviations used in this application. Otherabbreviations are as commonly used in the art.

                  TABLE 1                                                         ______________________________________                                        Abbreviations                                                                 ______________________________________                                        A    =     Ala = Alanine    M = Met = Methionine                              C    =     Cys = Cysteine   N = Asn = Asparagine                              D    =     Asp = Aspartic Acid                                                                            P = Pro = Proline                                 E    =     Glu = Glutamic Acid                                                                            Q = Gln = Glutamine                               F    =     Phe = Phenylalanine                                                                            R = Arg = Arginine                                G    =     Gly = Glycine    S = Ser = Serine                                  H    =     His = Histidine  T = Thr = Threonine                               I    =     Ile = Isoleucine V = Val = Valine                                  K    =     Lys = Lysine     W = Try = Tryptophan                              L    =     Leu = Leucine    Y = Tyr = Tyrosine                                Boc  =     tert-butyloxycarbonyl                                              CFU  =     colony forming unit                                                DFP  =     diisopropylfluorophosphate                                         HLE  =     human leukocyte elastase                                           Pam  =     (phenylacetamido) methyl                                           ______________________________________                                    

ED₅₀ is the concentration of an antimicrobial agent which kills (orotherwise inhibits growth) 50% of the input indicator microorganism orcell under particular test conditions.

For convenience, the peptides newly disclosed herein are named accordingto the amino acid positions in mature Cat G (FIG. 8) CG 1-20 representsamino acid residues 1-20 of the mature Cat G sequence and has thesequence IIGGRESRPHSRPYMAYLQI (SEQ ID NO:16).

CG 21-40 corresponds in sequence to amino acids 21-40 of Cat G,QSPAGQSRCGGFLVREDFVL (SEQ ID NO:17).

CG 41-60, corresponding to amino acids 41-60 of Cat G, has the sequenceTAAHCWGSNINVTLGAHNIQ (SEQ ID NO:18).

CG 61-80, corresponding to amino acids 61-80 of Cat G, has the sequenceRRENTQQHITARRAIRHPQY (SEQ ID NO:19).

CG 77-96, corresponding to amino acids 77-96 of Cat G, has the aminoacid sequence HPQYNQRTIQNDIMLLQLSR (SEQ ID NO:20).

CG 97-116, corresponding to amino acids 97-116 of Cat G, has thesequence RVRRNRNVNPVALPRAQEGL (SEQ ID NO:21).

CG 117-136, corresponding to amino acids 117-136 of Cat G, has thesequence RPGTLCTVAGWGRVSMRRGT (SEQ ID NO:22).

CG 137-156, corresponding to amino acids 137-156 of Cat G, has thesequence DTLREVQLRVQRDRQCLRIF (SEQ ID NO:23).

CG 157-176, corresponding to amino acids 157-176 of Cat G, has thesequence GSYDPRRQICVGDRRERKAA (SEQ ID NO:24).

CG 177-197, corresponding to amino acids 177-197 of Cat G, has thesequence FKGDSGGPLLCNNVAHGIVSY (SEQ ID NO:25).

CT 198-223, corresponding to amino acids 198-223 of Cat G, has thesequence GKSSGVPPEVFTRFVSSFLPWIRTTMR (SEQ ID NO:26).

Antimicrobial activity, as used herein, refers to the ability of apeptide of the present invention to kill at least one species selectedfrom the group consisting of Gram positive bacteria, Gram negativebacteria, fungi, and protozoans. It is increasingly preferred that thepeptide kill at least 50%, 60%, 70%, 80%, 90% or all cells of at lestone species of gram positive or gram negative bacteria, fungi, orprotozoans. Sensitive Gram positive bacteria can include, but are notlimited to, Staphylococcus aureus. Sensitive Gram negative bacteriainclude, but are not limited to, Escherichia coli, Neisseriagonorrhoeae, and Pseudomonas aeruginosa. Periodontal disease-associatedbacteria include Capnocytophaga sputigena, Actinobacillusactinomycetemcomitans and Eikenella corrodens. Capnocytophaga sputigenaATCC 33123 is sensitive to IIGGR (SEQ ID NO:1), IIGGRESRPHSRPYMAYLQI(SEQ ID NO:16) and HPQYNQ (SEQ ID NO:13). A. acetinomycetemcomitans issensitive to IIGGR (SEQ ID NO:1) and HPQYNQR (SEQ ID NO:3). E. corrodensis more sensitive to IIGGR (SEQ ID NO:1) than to HPQYNQR (SEQ ID NO:3).Sensitive fungi can include, but are not limited to, Candida albicans.Antimicrobial activity can also refer to the ability to kill or inhibitthe growth of other cells, in particular, those which are tumor cells orvirus-infected cells.

The antimicrobial peptides of the present invention are oligopeptideswhich possess antimicrobial activity, as defined herein. Theseantimicrobial peptides may contain modifications such as acetylation,provided that the antimicrobial activity is not destroyed. Chemicalmodifications which do not destroy antimicrobial activity are thosewhich do not substantially decrease the hydrophilicity of theantimicrobial peptide and those which are not bulky hydrophobic chemicalgroups, particularly for antimicrobial peptides related in sequence toHPQYNQR. Modified peptides with antimicrobial activity are functionallyequivalent to the antimicrobial peptides of the present invention. Suchmodified peptides with antimicrobial activity include, but are notlimited to, (1-methyl-H)QYNQR, (3-methyl-H)PQYNQR, (Ac--H)PQYNQR andHPAYNA^(M) K.

Antibacterial pharmaceutical compositions, as defined herein, comprise apharmaceutically acceptable carrier and one or more antibacterialpeptides of the present invention. Such antimicrobial pharmaceuticalcompositions may be formulated in ways, as understood in the art, foruse for topical application, for gingival application (for gingivitis orperiodontal disease) or for local or systemic injection. For use in thetreatment or prevention of gingivitis, the peptides of the presentinvention can be incorporated in effective amounts in a dental rinse forapplication to the buccal area, or they may be incorporated in othersuitable compositions for topical application. The antibacterialpeptides of the present invention may also be incorporated in effectiveamounts in chewing gum, lozenges for sucking, toothpowder or toothpaste.The antibacterial peptides of the present invention can comprise from0.001% to 50% by weight of such compositions. It will be understood thata composition for systemic injection will contain an antimicrobialpeptide, e.g., an antibacterial peptide such as HPQYNQR, in atherapeutically effective amount or a therapeutically effective amountof an antimicrobial peptide can be conjugated to an antibody, or anyother compound as understood in the art, with specificity for the targetcell type. The choice of the peptide will be made with consideration ofimmunogenicity and toxicity to the infected host, effective dose of thepeptide, and the sensitivity of the target microbe to the peptide, aswell-understood in the art.

The Cat G protein was analyzed to determine whether the same portions ofthe protein were responsible for the enzymatic and antibacterialactivity. Human Cat G was purified and digested with the proteolyticenzyme clostripain. Peptides resulting from that digestion were purifiedand individually tested for antibacterial and enzymatic activity. Noneof the peptides tested exhibited the chymotrypsin-like activity of theintact molecule. However, two Cat G-derived peptides exhibitedantibacterial activity using Staphylococcus aureus or Neisseriagonorrhoeae as the indicator organism. Those peptides were IIGGR (SEQ IDNO:1) (peptide 1; amino acids 1-5) and HPQYNQR (peptide 2; amino acids77-83) (SEQ ID NO:3). Similar antibacterial activities were observed forsynthetic peptides identical in sequence to the above-noted peptides.Similarly, the oligopeptide corresponding in amino acid sequence toamino acids 1-20 of Cat G (i.e., SEQ ID NO:16) exhibited strongbactericidal activity against Pseudomonas aeruqinosa. Even moreeffective as an antimicrobial agent is the oligopeptide corresponding insequence to amino acids 1-20 of Cat G, but which retains some blockinggroups from the component derivatized amino acids in chemical peptidesynthesis or an artifactual reaction product. The chemical identity ofthe substituents on that oligopeptide or reaction product have not yetbeen identified. FIG. 4(A) illustrates killing of S. aureus. and N.gonorrhoeae by these two peptides.

A peptide with a one amino acid substitution (V for I at position 2 ofIIGGR (SEQ ID NO:1)) was synthesized and tested for antibacterialactivity. This sequence is identical with the first five amino acids ofhuman leukocyte elastase, CAP37 and azurocidin. Killing of both the Grampositive and the Gram negative test organism was less efficient with theIVGGR (SEQ ID NO:2) sequence (ED₅₀ >8.75×10⁻⁴ M) than with the IIGGRsequence (ED₅₀ =4.3×10⁻⁴ M), although the IVGGR sequence was somewhatmore effective against gonococci than staphylococci (see FIG. 3A).

Killing of staphylococci and gonococci by HPQYNQR (SEQ ID NO:3) andAPQYNQR (SEQ ID NO:27) was also tested. The results are shown in FIG.3(B); for HPQYNQR (SEQ ID NO:3) the ED₅₀ =1.19×10⁻⁴ M for S. aureus, andthe ED₅₀ for N. gonorrhoeae was 5.0×10⁻⁵ M. The replacement of histidinewith alanine (APQYNQR) abolished its bactericidal activity for bothmicroorganisms.

Antimicrobial activity of the Cat G-derived peptides was found to bedependent on assay temperature, pH and ionic strength. As forfull-length Cat G, effective killing of S. aureus by the syntheticpeptides was optimal at 37° C. with activity from about 30° to about 39°C., and was optimal at pH values of 7.0-7.5 for the range of pH valuestested (Results for HPQYNQR (SEQ ID NO:3) are shown in FIGS. 4 and 6B).The antimicrobial action of these peptides was optimal in standard HBSS(0.12M NaCl); conditions of increased ionic strength (0.17-0.21M NaCl)completely inhibited the antibacterial action (FIG. 6B) except forpeptides HPQYNPK (SEQ ID NO:49) or Ac-HPQYNQR.

Because the peptides tested individually showed significantly lessactivity than the natural Cat G protein or the clostripain digest, IIGGR(SEQ ID NO:1) and HPQYNQR (SEQ ID NO:3) were tested for synergisticactivity. Under optimal conditions of temperature, pH and ionicstrength, IIGGR and HPQYNQR together were about twice as active againstS. aureus than when tested individually, but still less active thannative Cat G or a clostripain digest of Cat G. A synthetic peptideconsisting of the peptides 1 and 2 joined by a bridge (AIR) had activitysimilar to that of HPQYNQR alone (ED₅₀ =5.0×10⁻⁵ M). Thus, these twopeptides together were less effective against S. aureus than natural CatG (ED₅₀ =4.0×10⁻⁶ M), suggesting that other antimicrobial domains orsequences flanking peptides 1 and 2 in the full-length moleculecontribute to the total antimicrobial activity of full-length Cat G.Alternatively, other peptides in the clostripain-Cat G digestion mixturemay have potentiated the activity of IIGGR (SEQ ID NO:1) and HPQYNQR(SEQ ID NO:3).

The antibacterial activities of IIGGR (SEQ ID NO:1) and HPQYNQR (SEQ IDNO:3) were compared with other known antimicrobial agents (FIG. 5).These peptides exhibited activities roughly comparable to those of apool of defensins and to the clinically useful antibioticschloramphenicol and tetracycline (test concentration of each compound5.0×10⁻⁴ M). For the heptapeptides, this corresponds to about 100micrograms per ml. Chloramphenicol was more effective at thisconcentration than the defensin pool or the test peptides, and thesepeptides from Cat G were more active than tetracycline, streptomycin,kanamycin or the defensin pool.

IIGGR (SEQ ID NO:1) and HPQYNQR (SEQ ID NO:3) were used to search knownprotein sequences of other cytotoxic serine proteases present in thecytolytic lymphocytes of mice and humans for partial amino acid sequenceidentities. Proteins containing the IIGGR sequence at the N-terminiinclude Cat G and eosinophil cationic protein (Gleich et al. (1986)Proc. Natl. Acad. Sci. USA 83:3146-3150). Human leukocyte elastase(Gabay et al. (1989) supra; azurocidin (Sinha et al. (1987) Proc. Natl.Acad. Sci. USA 84:2228-2232); and CAP 37 (Pereira et al. (1990) J. Clin.Invest. 85:1468-1476) begin with the sequence IVGGR (SEQ ID NO:2). Humanleukocyte elastase does not have antimicrobial activity in vitro. CCCP Iand human lymphocyte granzymes B, D, E and G begin with IIGGH (SEQ IDNO:28) while RMCP II begins with the sequence IIGGV (SEQ ID NO:29)(Salvesen et al. (1987) supra). It may be assumed that the IIGGR (SEQ IDNO:1) of Cat G is located on the interior of the protein because itforms a salt bridge with Asp-170, a residue which has been invariablyidentified to be a part of the binding site of other chymotrypsin-likeserine proteases. This Cat G residue corresponds to Asp-102 in thecatalytic triad of chymotrypsin. This hypothesis is consistent with theX-ray crystallographic structure of chymase (Remington et al. (1988)Biochemistry 27:8097-8115), which has significant sequence similaritywith Cat G; a similar domain is buried in the interior of the molecule.It is probable that HPQYNQR is oriented toward the surface of Cat Gbecause of the positioning of an analogous sequence in chymase, thestructure of which has been predicted.

Human granzymes A and B (granzyme B is also known as human lymphocyteprotease) both contain internal sequences related to HPQYNQR (SEQ IDNO:3). HPAYNPK (SEQ ID NO:5), corresponding to an internal region ofhuman leukocyte protease, possesses broad spectrum antimicrobialactivity in vitro. The related sequences of mouse granzymes A, C, E, Fand G do not possess such activity (see Table 5).

Variant peptides related to the sequences IIGGR and HPQYNQR were testedfor antimicrobial activity. The results of testing variant sequences ofthe HPQYNQR peptide sequence for antimicrobial activity has led to theformulation of a consensus sequence for an antimicrobial peptide: HX₁ X₂X₃ X₄ X₅ X₆ (SEQ ID NO:4).

Preferably X₁ is proline, alanine or histidine; X₂ is one of asparagine,aspartic acid, glutamine, glutamic acid or alanine; X₃ is tyrosine orphenylalanine; X₄ is asparagine or alanine; X₅ is proline, glutamine,alanine or N-methyl alanine; and X₆ is lysine, arginine, alanine, --OHor --NH₂.

Antimicrobial hexa- and heptapeptide sequences falling within theconsensus sequence include, but are not limited to, HPQYNQR (SEQ IDNO:3), HPAYNPK (SEQ ID NO:5), HPAYNPR (SEQ ID NO:6) and HPQYNQR (SEQ IDNO:3). Additional antimicrobial peptide sequences related to HPQYNQRinclude, but are not limited to, HPQYAQR (SEQ ID NO:8), HPQYNQA (SEQ IDNO:9), HPQYNAR (SEQ ID NO:10), HPQYNPR (SEQ ID NO:30), HAQYNQR (SEQ IDNO:11), HPQYNQ (SEQ ID NO:12), HHQYNQR (SEQ ID NO:31) and HPQYNQ^(M) K.Preferably, the oligopeptides consist essentially of the foregoingsequences. CG 77-96 (HPQYNQRTIQNDIMLLQLSR) (SEQ ID NO:14) does notexhibit antimicrobial activity.

To determine the amino acids essential for the broad spectrum activityof HPQYNQR (SEQ ID NO:3), synthetic peptides with single alaninesubstitutions were prepared and tested for their antibacterial activityin vitro. The parental peptide sequence HPQYNQR and the followingalanine-substituted derivatives (HPAYNQR (SEQ ID NO:7), HPQYAQR (SEQ IDNO:8) and HPQYNQA) (SEQ ID NO:9) exerted antibacterial action in vitroagainst S. aureus strain 8325-4 at a concentration of about 5×10⁻⁴ M,while certain other alanine-substituted derivatives (HPQYNAR (SEQ IDNO:10) and HPAYNPR) (SEQ ID NO:6) had reduced antibacterial activity,and still others (APQYNQR (SEQ ID NO:27) and HPQANQR (SEQ ID NO:32)) hadno antibacterial activity in this assay. Similar activities or lack ofactivity was also observed with E. coli and with N. gonorrhoeae. Theseresults are given in Table 2.

                  TABLE 2                                                         ______________________________________                                        Bactericidal Action of Synthetic Peptide                                      Variants of Antimicrobial HPQYNQR                                             Peptide Secquence.sup.1                                                                         % Survival of S. aureus                                                                       N                                           ______________________________________                                        HPQYNQR (SEQ ID NO: 3)                                                                          28.6 ± 1.57  15                                          APQYNQR (SEQ ID NO: 27)                                                                         98.5 ± 2.1   12                                          HAPYNQR (SEQ ID NO: 11)                                                                         31.2 ± 2.4   9                                           HPAYNQR (SEQ ID NO: 7)                                                                          43.6 ± 2.54  6                                           HPQANQR (SEQ ID NO: 32)                                                                         96.7 ± 4.34  6                                           HPQYAQR (SEQ ID NO: 8)                                                                          30.6 ± 0.85  6                                           HPQYNAR (SEQ ID NO: 10)                                                                         51.2 ± 3.6   6                                           HPQYNQA (SEQ ID NO: 9)                                                                          39.8 ± 1.96  6                                           HPQKNTY (SEQ ID NO: 33)                                                                         98.6 ± 2.13  3                                            .sup.1 Antimicrobial synthetic peptide HPQYNOR, its derivatives with          single alanine substitutions at each position, and a control peptide          (HPQKNTY) were synthesized on an Applied Biosystem Model 403A peptide         synthesizer (0.1 mmol scale) using phenylacetamidomethyl or                   pmethylbenzyhydrylamine copoly (styrene/divinyl benzene) resins (Applied      Biosystems, Inc.) and tertbutyloxycarbonyl (Boc)protected amino acids. Th     data are presented as % survival ± SEM and represent results from at       least 3 separate experiments for each peptide with S. aureus as the           indicator organism.                                                      

These results suggest that the N-terminal histidine and the tyrosineresidue at position 4 may be important determinants for theantibacterial activity of HPQYNQR (SEQ ID NO:3). To more closely examinethe structural requirements of this peptide sequence needed formicrobicidal activity, several additional peptides (see Table 4)containing alterations in either His-1 or Tyr-4 and a truncated peptidelacking Arg-7 were prepared and tested for killing of the S. aureusindicator organism. Methylation of nitrogen at position 1 or 3 of theimidazole ring of His-1 had little effect on the antibacterial activity,but derivatives with the imidazole modified at nitrogen-3 with morebulky benzyl or dinitrophenyl groups were inactive. Moreover, asynthetic peptide containing the D-stereoisomer of His was alsoinactive. Thus it appears that major modifications of the imidazole ringof His-1 abolish antibacterial activity, perhaps due to impairedhydrogen bonding with other amino acid side chains. While replacement ofTyr-4 by alanine inhibited antibacterial activity (Table 2), partialactivity was observed where phenylalanine was present as the fourthresidue, suggesting that an aromatic amino acid at position 4 iscritical to antibacterial activity. The truncated pentapeptideHPQYN-amide was inactive, while the hexapeptide HPQYNQ-amide hadantibacterial activity, suggesting that at least six amino acid residuesare necessary to form a structure effective for antimicrobial activity.(See Table 3.) Surprisingly, an oligopeptide of the sequenceHPQYNQRTIQNDIMLLQLSR (SEQ ID NO:14) did not exhibit significantantimicrobial activity against either S. aureus or P. aeruginosa.

                  TABLE 3                                                         ______________________________________                                        Bactericidal Action of Synthetic Derivatives                                  of Antimicrobial Peptide HPQYNQR                                              Peptide Sequence.sup.1                                                                          % Survival of S. aureus                                                                       N                                           ______________________________________                                        HPQYNQR (SEQ ID NO: 3)                                                                          28.6 ± 1.57  15                                          APQYNQR (SEQ ID NO: 27)                                                                         98.5 ± 2.1   12                                          H.sup.1m QYNQR    37.3 ± 2.25  3                                           H.sup.3m PQYNQR.sup.2                                                                           18.3 ± 3.82  3                                           H.sup.Dnp PQYNQR.sup.2                                                                          102.2 ± 2.92 8                                           H.sup.Benzyl PQYNQR.sup.2                                                                       99.0 ± 2.6   6                                           .sup.Ac HPQYNQR   31.0 ± 1.6   5                                           .sup.Hep HPQYNQR  97.0 ± 2.8   3                                           D-HPQYNQR         98.6 ± 1.6   5                                           HPAYNQR (SEQ ID NO: 7)                                                                          43.6 ± 2.54  6                                           HPQANQR (SEQ ID NO: 32)                                                                         96.7 ± 4.34  6                                           HPQFNQR (SEQ ID NO: 34)                                                                         45.0 ± 4.91  6                                           HPQYNQA (SEQ ID NO: 9)                                                                          39.8 ± 1.96  6                                           HPQYNQ-amide      32.8 ± 1.83  3                                           HPQYN-amide       98.5 ± 2.85  5                                           ______________________________________                                         .sup.1 All peptides were tested at 5 × 10.sup. -4 M as described in     Table 2.                                                                      .sup.2 Modified histidines are 1methyl (1m), 3methyl (3m),                    2,4dinitrophenyl (Dnp), benzyl, acetyl (Ac), and heptanoyl (Hep), and D       stereoisomer (DHPQYNQR) as described in the text.                        

To learn whether the hydrophilic nature of HPQYNQR is an importantfeature for antibacterial action, derivatives containing an acetyl or anheptanoyl moiety attached to the N-terminal amino group were synthesizedand tested. The small increase in hydrophobicity imparted by the acetylgroup has no significant effect, while the more bulky and hydrophobicheptanoyl group rendered the HPQYNQR (SEQ ID NO:3) derivative inactive.

Peptides corresponding or related to sequences within mouse granzymes A,B, C, D, E, F and G and human granzymes A and B were synthesized. Asshown in Table 4, only the synthetic peptides corresponding to mousegranzyme B and human granzyme B had some antibacterial activity.

The human granzyme B-derived peptide differs from the Cat G peptide atpositions 3, 6 and 7. To test how these differences affectedantibacterial activity, variants of the Cat G and granzyme B peptidescontaining some of these heterologous amino acids or with alaninesubstitution at position 3 or 6 were synthesized and tested (Table 5).Placement of the Ala-3 residue in the Cat G only had a slight effect onantibacterial action (Tables 2 and 4). Conversely, placement of theGln-3 residue in the Granzyme B peptide severely inhibited antibacterialactivity. The Pro-6 residue of the granzyme B peptide also appeared tobe crucial, because the replacement of Pro-6 with alanine abolishedmicrobicidal activity. To further test the importance of Pro-6, aderivative containing N-methylalanine, which lacks the ring structure ofproline but might mimic its hydrogen bonding potential, was synthesizedand found to have antibacterial activity. The contribution of the Pro-6residue to antibacterial activity in the granzyme B-derived peptideappeared to be unique for this peptide, because substitution at position6 in the Cat G-derived peptide suppressed antibacterial activity.Further derivatives of HPQYNQR (SEQ ID NO:3) with amino acidreplacements at the third position were synthesized and tested forantimicrobial activity. HPNYNQR (SEQ ID NO:35) and HPEYNQR (SEQ IDNO:36) had antimicrobial activity comparable to that of HPAYNQR (SEQ IDNO:37) while HPLYNQR (SEQ ID NO:37) lacked activity in the assay. (Table4) HPDYNQR (SEQ ID NO:53) will have activity comparable to that ofHPAYNQR (SEQ ID NO:7).

                  TABLE 4                                                         ______________________________________                                        Activity of Cat G Peptides with Third Position Replacements                   Peptide            Percent Survival                                           ______________________________________                                        HPQYNQR (SEQ ID NO: 3)                                                                           21.2%                                                      HPAYNQR (SEQ ID NO: 7)                                                                           40.3%                                                      HPEYNQR (SEQ ID NO: 36)                                                                          45.3%                                                      HPNYNQR (SEQ ID NO: 35)                                                                          42.9%                                                      HPLYNQR (SEQ ID NO: 37)                                                                          100.8%                                                     ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Bactericidal Action of Synthetic Human                                        and Mouse Granzyme Peptides.sup.1                                                                        % Survival                                         Peptide       Sequence     of S. aureus                                                                             N                                       ______________________________________                                        Cat G         HPQYNQR       28.6 ± 1.57                                                                          15                                                    (SEQ ID NO: 3)                                                  Cat G (Pro-6) HPQYNPR      78.7 ± 2.8                                                                            4                                                     (SEQ ID NO: 50)                                                 Human granzyme A                                                                            YPCYDPA       102 ± 2.6                                                                            3                                                     (SEQ ID NO: 51)                                                 Human granzyme B                                                                            HPAYNPK        36 ± 2.5                                                                            9                                                     (SEQ ID NO: 5)                                                  Human granzyme B'.sup.2                                                                     HPAYNAK        97 ± 1.8                                                                            3                                                     (SEQ ID NO: 52)                                                 Human granzyme B''                                                                          HPQYNPK      95.1 ± 3.4                                                                            5                                                     (SEQ ID NO: 53)                                                 Human granzyme B'''                                                                         HPAYNPR      87.5 ± 4.3                                                                            5                                                     (SEQ ID NO: 6)                                                  Human granzyme B''''                                                                        HPAYNA.sup.m K.sup.3                                                                       40.5 ± 1.6                                                                            3                                       Human granzyme B'''''                                                                       HPAYNP-amide 42.5 ± 3.6                                                                            5                                       Mouse granzyme A                                                                            YPCYDEY      99.5 ± 1.7                                                                            3                                                     (SEQ ID NO: 54)                                                 Mouse granzyme B                                                                            HPDYNPK        62 ± 3.4                                                                            3                                                     (SEQ ID NO: 55)                                                 Mouse granzyme C                                                                            HPDYNPD       104 ± 3.8                                                                            3                                                     (SEQ ID NO: 56)                                                 Mouse granzyme D,E.sup.2                                                                    HPDYNAT        98 ± 2.7                                                                            3                                                     (SEQ ID NO: 57)                                                 Mouse granzyme F                                                                            HPAYDDK        98 ± 1.9                                                                            3                                                     (SEQ ID NO: 58)                                                 Mouse granzyme G                                                                            HPAFDRK       102 ± 2.8                                                                            3                                                     (SEQ ID NO: 59)                                                 ______________________________________                                         .sup.1 All peptides were tested at 5 × 10.sup.4 M as described in       Table 2.                                                                      .sup.2 Peptide sequences for mouse granzymes D and E are identical. Human     granzyme B variants are designated B'-B'''''.                                 .sup.3 A.sup.M, Nmethyl alanine.                                         

While the deletion of the C-terminal lysine-7 residue from HPAYNPK (SEQID NO:5) had no effect on the bactericidal action of the granzymeB-related peptide, its replacement with arginine as in the Cat G peptideseverely inhibited antibacterial activity.

As for the full-length Cat G, the antibacterial action of the granzymeB-derived peptide was found to be optimal at slightly basic pH (FIG. 6A)although, like the acetylated derivative of HPQYNQR, significantantibacterial activity was observed at a pH of 6.0. As for full-lengthCat G, the antibacterial action of human granzyme B was also sensitiveto increasing concentrations of NaCl in the incubation mixture.Surprisingly, the Cat G-derived peptide HPQYNQR (SEQ ID NO:3) and itsacetylated derivative retained antibacterial activity even at thehighest NaCl concentration (0.21M) tested (FIG. 6B).

As seen with the Cat G-derived peptide, the antibacterial action ofhuman granzyme B extended to E. coli and N. gonorrhoeae as well as S.aureus. Killing of the S. aureus indicator was more rapid with the CatG-derived peptide than with the human granzyme B-derived peptide (FIG.6C).

Results with variant peptides related to HPQYNQR (SEQ ID NO:3) suggestthat the N-terminal histidine and the internal tyrosine residues wereimportant in generating antimicrobial activity. A substitution ofalanine for histidine, D-histidine for the L-isomer or the attachment ofa 2,4-dinitrophenyl group to the imidazole group of L-histidine resultedin a loss of activity. Similarly, a substitution of alanine for tyrosineat position 4 led to a loss of microbicidal activity (see Table 4).

To determine whether hydrophilicity of the antimicrobial peptide wasdeterminant of activity, HPQYNQR (SEQ ID NO:3) was modified by theaddition of an acetyl or a heptanoyl group to the N-terminal aminogroup. Acetylation did not affect activity, while the heptanoylderivative lacked antimicrobial activity.

The antibacterial activity of the HLP-derived peptide (HPAYNPK (SEQ IDNO:5)) cannot be readily explained using the Cat G sequence (HPQYNQR(SEQ ID NO:3)) as a canonical model. The alanine residue at position 3and the proline at position 6 of the HLP-derived peptide cannot resultfrom conservative amino acid changes in HPQNQR, although the lysine forarginine at position 7 is a conservative change. The ability of aposition 7 substitution variant (alanine for arginine) of the Cat Gsequence to retain significant killing activity might suggest that thisis not a key position in determining activity. An alanine substitutionat position 3 (for glutamine) decreased activity of the Cat G-relatedpeptide.

The result of testing variant sequences of the HPQYNQR peptide sequencefor antimicrobial activity has led to the formulation of a consensussequence for an antimicrobial peptide. HX₁ X₂ X₄ X₅ X₆, where X₁ isproline, histidine or alanine; X₂ is aspartic acid, asparagine, glutamicacid, glutamine, alanine, serine, threonine, isoleucine, valine,histidine, tyrosine, arginine, methionine oxide or methionine sulfone;X₃ is tyrosine, phenylalanine, tryptophan or naphthyl-alanine; X₄ isasparagine or alanine; X₅ is glutamine, proline, alanine, or N-methylalanine; and X₆ is arginine, lysine, alanine, --OH or --NH₂ (SEQ IDNO:4).

Cat G-derived peptides were tested for antimicrobial activity againstCapnocytophaga sputigena ATCC 33123, which is the same as that nowavailable as C. sputigena ATCC 33612 (American Type Culture Collection,Rockville, Md.). C. sputigena is representative of oral pathogensassociated with periodontal disease and/or gingivitis. As shown in FIG.7, IIGGR (SEQ ID NO:1) and CG 1-20 (SEQ ID NO:16) are effective againstC. sputigena ATCC 33123 in vitro. When incorporated in pharmaceuticalcompositions, one or more of the peptides related in sequence to Cat Gcan be used to ameliorate gingivitis and/or treat periodontal disease.Compositions for oral use include oral rinses, lozenges and formulationsfor topical application to the gums. The skilled artisan can use theteachings of the present specification and knowledge readily accessibleto the art to prepare pharmaceutically useful formulations for oralapplication, topical or other applications, particularly after animalstudies to confirm that these peptides are not toxic to the human oranimal host and are effective in vivo.

Because the antimicrobial activity resulting from the IIGGR and HPQYNQRpeptides recovered after clostripain digestion was less than 1% of theactivity of intact Cat G, an alternate approach was pursued. Elevenpeptides that span the entire 223 amino acid cathepsin G protein weresynthesized. These eleven peptides were tested for antibacterial actionagainst N. gonorrhoeae, P. aeruginosa and S. aureus. Of the elevenpeptides, only the peptide corresponding to residues 117-136 in thefull-length cathepsin G (CG 117-136) (SEQ ID NO:22) displayedantibacterial action against all three pathogens; 500 μg of peptide perml killed 5-6 logs of P. aeruginosa and S. aureus. See Table 6 for thepeptide sequences and activities, as measured using a crude peptideconcentration of 500 μg/ml with an input viable cell concentration ofabout 10⁷ colony forming units/ml (CFU/ml) in 1/100 strength HBSS.CB117-136 will be useful as an antibacterial agent against a wide rangeof bacteria, including pathogens.

                                      TABLE 6                                     __________________________________________________________________________    Antibactericidal Activity of Crude Peptides.sup.1                                                  Log Kill (500 μg/ml)                                                       P. aeruginosa                                                                        N. gonorrhoeae                                    Peptide Sequence     ATCC 27853                                                                           strain WS1                                        __________________________________________________________________________    1             20                                                              IIGGRESRPHSRPYMAYLQI 5.507   2.57 (SEQ ID NO: 16)                             21            40                                                              QSPAGQSRCGGFLVREDFVL -0.034  0.05 (SEQ ID NO: 17)                             41            60                                                              TAAHCWGSNINVTLGAHNIQ 0.5    -0.09 (SEQ ID NO: 18)                             61            80                                                              RRENTQQHITARRAIRHPQY 5.15      0 (SEQ ID NO: 19)                              77            96                                                              HPQYNQRTIQNDIMLLQLSR 0.06    0.24 (SEQ ID NO: 20)                             97            116                                                             RVRRNRNVNPVALPRAQEGL 5.93   -0.21 (SEQ ID NO: 21)                             117           136                                                             RPGTLCTVAGWGRVSMRRGT 5.88    2.38 (SEQ ID NO: 22)                             137           156                                                             DTLREVQLRVQRDRQCLRIF 0.01    0.43 (SEQ ID NO: 23)                             157           176                                                             GSYDPRRQICVGDRRERKAA 1.02   -0.18 (SEQ ID NO: 24)                             177           197                                                             FKGDSGGPLLCNNVAHGIVSY                                                                              -0.79   0.68 (SEQ ID NO: 25)                             198           223                                                             GKSSGVPPEVFTRFVSSFLPWIRTTMR                                                                        5.73    0.78 (SEQ ID NO: 26)                             Buffer               -0.139  0.07                                             __________________________________________________________________________     .sup.1 Peptides shown in Bold were purified by RPHPLC and tested vs. P.       aeruginosa, N. gonorrhoeae and S. aureus (see Table 7A).                 

Table 7A provides antimicrobial activities of peptides purified byRP-HPLC, determined as above. Only CG 117-136 (SEQ ID NO:22) exhibitshigh activity against P. aeruqinosa, S. aureus and N. gonorrhoeae. Table7B discloses the ED₉₀ in μg/ml for the antibacterial peptides, asmeasured with P. aeruqinosa ATCC 27853 as above. ED₉₀ is the doserequired to kill 90% of the input cells in 2 h at 37° C. in 1/100strength HBSS, where the input viable cell concentration is about 10⁷CFU/ml.

                                      TABLE 7A                                    __________________________________________________________________________    Antibacterial Action of RP-HPLC-Purified Peptides                                     Log Kill (500 μg/ml                                                        P. aeruginosa                                                                         N. gonorrhoeae                                                                        S. aureus                                             Peptide ATCC 278533                                                                           strain WSI                                                                            strain 8325-4                                         __________________________________________________________________________    CG 1-20 1.0     0.43     0.3 (SEQ ID NO: 16)                                  CG 61-80                                                                              5.15.sup.1                                                                            ND       0.58 (SEQ ID NO: 19)                                 CG 97-116                                                                             0.22    0.2     -0.1 (SEQ ID NO: 21)                                  CG 117-136                                                                            5.88.sup.1                                                                            4.20     5.34 (SEQ ID NO: 22)                                 CG 198-223                                                                            5.73.sup.1                                                                            ND       0.26 (SEQ ID NO: 26)                                 Buffer Control                                                                        -0.139  -0.07   -0.25                                                 __________________________________________________________________________     .sup.1 Also active against four clinical isolates of P. aeruginosa            .sup.2 Not determined                                                    

                  TABLE 7B                                                        ______________________________________                                        Potency of Synthetic Cathepsin G Peptides Against                             P. aeruginosa                                                                 Synthetic Peptides                                                                              ED.sub.90 (μg/ml)                                        ______________________________________                                        CG 1-20            500 (SEQ ID NO: 16)                                        CG 61-80            75 (SEQ ID NO: 19)                                        CG 97-116         >500 (SEQ ID NO: 21)                                        CG 117-136          15 (SEQ ID NO: 22)                                        CG 198-223         115 (SEQ ID NO: 26)                                        ______________________________________                                    

It was noted that CG 1-20 (SEQ ID NO:16) and CG 97-116 (SEQ ID NO:21)were highly active as crude peptides, but exhibited much lower activitywhen purified by RP-HPLC. Without wishing to be bound by any particularhypothesis, the inventors suggest that the high activity in the crudepeptide preparation is due to one or more residual blocking orsubstituent groups or some unidentified side reaction product generatedduring the hydrogen fluoride cleavage and/or post-synthetic work-up.

Those peptides which exhibited significant activity against P.aeruginosa ATCC 27853, were also tested against four independentclinical isolates. P. aeruginosa ATCC 27853 (American Type CultureCollection, Rockville, Md.) is the strain used for testing antimicrobialactivity against P. aeruginosa unless otherwise noted. It is astandardized strain for antibiotic-susceptibility testing ofpseudomonads (see, e.g., Code of Federal Regulations, Title 21, Part460, 1987). CG 61-80 (SEQ ID NO:19) and CG 198-223 (SEQ ID NO:26exhibited some variability in effectiveness for killing of clinicalstrains, but so far as tested, CG 117-136 (SEQ ID NO:22) appeared to bea highly effective bactericidal peptide for P. aeruqinosa as well as N.gonorrhoeae and S. aureus (see Tables 7A, 8). Agents effective againstP. aeruginosa are needed in the art because multiple antibioticresistance is quite common among clinical strains, and thereforeresultant infections are often difficult to treat.

                  TABLE 8                                                         ______________________________________                                        Bactericidal Activity of HPLC-Purified Peptides                               Against P. aeruginosa Clinical Isolates                                                    Log Reduction in Viability.sup.1                                 P. aeruginosa                                                                           Control  CG 61-80  CG 117-136                                                                            CG 198-223                               ______________________________________                                        ATCC 27853                                                                              -0.14    5.49      5.95    5.35                                     #385128   -1.1     1.91      6.17    4.71                                     #36152    -0.89    4.86      5.38    4.28                                     #27853    -0.58    5.80      5.79    4.80                                     #A-91-330-0347                                                                          -0.41    4.11      6.40    1.17                                     ______________________________________                                         .sup.1 3-5 × 10.sup.7 CFU/ml of the designated strains were exposed     to 500 μg/ml of HPLCpurified peptides in 1/100 strength BHSS (10 mm        sodium phosphate, pH 7.0, 10 mM NaCl.                                    

To further characterize the activity of CG 117-136, a "D-enantiomer,"composed only of D-amino acids but in the same sequence, was synthesizedand tested for antimicrobial activity. The L- and D-forms of this sameamino acid sequence had equivalent bactericidal activity against both P.aeruginosa and N. gonorrhoeae (Table 9). This result suggests thatkilling does not require the recognition of a microbial target with achiral center.

                  TABLE 9                                                         ______________________________________                                        The Antibacterial Capacity of CG 117-136 (SEQ ID NO: 22)                      is Independent of Stereochemistry                                                         Log Kill.sup.1                                                                  P. aeruginosa                                                                            N. gonorrhoeae                                       Peptide       ATCC 27853 strain WS1                                           ______________________________________                                        L-enantiomer  5.62       4.20                                                 D-enantiomer  5.92       4.20                                                 ______________________________________                                         .sup.1 P. aeruqinosa (3 × 10.sup.7 CFU/ml) was exposed to 125           μg/ml of each peptide, while N. gonorrhoeae (5 × 10.sup.6 CFU/ml     was exposed to 500 μg/ml of each peptide.                             

As shown in FIG. 2B, CG 117-136 (SEQ ID NO:22) is predicted to have ahydrophobic domain in the N-terminal portion of the peptide and acationic, hydrophilic domain in the C-terminal portion. Thecontribution(s) of these domains to antimicrobial activity was assessedby synthesizing truncated versions of CG 117-136. Both domains wererequired for full activity. Omission of either C-or N-terminal residuesdestroyed activity against S. aureus, while omission of the fiveN-terminal residues resulted in only about a 10-fold drop in activityfor both P. aeruginosa and N. gonorrhoeae. Omission of the tenN-terminal residues caused nearly total loss of activity against P.aeruqinosa and a lesser reduction in activity as measured against N.gonorrhoeae (see Table 10A, 10B).

                  TABLE 10A                                                       ______________________________________                                        Summary of Domains in CG 117-136 and in                                       Truncated CG 117-136 variants                                                                   Hydrophobic                                                                              Hydrophilic                                      Peptide           Domain     Domain                                           ______________________________________                                        CG 117-136 (SEQ. ID. NO: 22)                                                                    +          +                                                CG 117-129 (SEQ. ID. NO: 38)                                                                    +          -                                                CG 122-136 (SEQ. ID. NO: 39)                                                                    ±       +                                                CG 127-136 (SEQ. ID. NO: 40)                                                                    -          +                                                ______________________________________                                    

                  TABLE 10B                                                       ______________________________________                                        Antibacterial Action of CG 117-136 and                                        Truncated Variants of CG 117-136                                                        Log Kill (500 μg/ml                                                                                 S. aureus                                              P. aeruginosa                                                                            N. gonorrhoeae                                                                            strain                                     Peptide     ATCC 27853 strain WS1  8325-4                                     ______________________________________                                        CG 117-136  5.8        4.20        5.34                                       (SEQ. ID NO: 22)                                                              CG 117-129  0.86       0.05        0                                          (SEQ. ID NO: 38)                                                              CG 122-136  4.75       4.18        0                                          (SEQ. ID NO: 39)                                                              CG 127-136  0.27       3.20        0                                          (SEQ. ID NO: 40)                                                              ______________________________________                                    

The secondary structure of CG 117-136 (SEQ ID NO:22) has been predictedby computer analysis to exhibit β-Sheet structure. By contrast, severalpeptides known to interrupt the gram-negative envelope are α-helicalpeptides (See, e.g., Vaara, M. (1992) Microbiological Rev. 56:395-411).

The potent antibacterial peptide CG 117-136 (SEQ ID NO:22) displayspartial significant sequence identities with other serine proteases orserine protease-like proteins (FIG. 10), termed serpocidins, based ontheir toxic action against bacteria and eucaryotic cells. The mechanismof cytotoxicity has not been defined, but it is likely that theserpocidins behave as membrane-disorganizing agents. Electronmicroscopic analysis of CG 117-136-treated P. aeruginosa cells revealedsimilar effects on cell morphology to those seen after treatment withpolycationic agents. Lysis did not result from CG 117-136 treatment.

A comparison of the amino acid sequence of CG 117-136 (SEQ ID NO:22) topartial sequences of other antimicrobial proteins and serine proteasesis given in FIG. 10. The dots represent amino acid identity. The skilledartisan can readily identify variants of the CG 117-136 sequence (or ofother antibacterial peptides disclosed herein) by synthesizing definedvariants and testing as taught herein.

The sensitivities of the P. aeruginosa and S. aureus indicator cells toenzymatically-active and inactive Cat G were determined (see Table 11).Surprisingly, peptides (CG 61-80, CG 117-136, CG 198-223) (SEQ ID NO:19,21, 26, respectively) from cathepsin G display potent bactericidalaction in vitro even against a pathogen (P. aeruginosa) that is killedonly by enzymatically active Cat G.

                  TABLE 11                                                        ______________________________________                                        Bactericidal Capacities of Enzymatically-Active                               and -Inactive Cathepsin G                                                                       Log Reduction in CFU/ml.sup.1                                                 S. aureus                                                                            P. aeruginosa                                        ______________________________________                                        Control             -0.18    -0.76                                            50 μg/ml enzymatically                                                                         2.82     2.41                                             active Cat G                                                                  50 μg/ml DFP-treated Cat G                                                                     2.93     0.125                                            ______________________________________                                         .sup.1 Approximately 5 × 10.sup.6 CFU/ml of the test bacteria were      incubated with the cathepsin G samples in 1% (w/v) TSB (tryticase soy         broth) for 2 h at 37° C. The results are average values from 2         determinations for each strain and preparation of cathepsin G.           

Only enzymatically-active cathepsin G kills P. aeruqinosa, while otherpathogens are readily killed by both active and inactive cathepsin G. Itis theorized that in order for the antibacterial peptides to properlyinteract with the pseudomonad cell envelope, the structure of the activesite must be in its native state in order to allow accessibility ofbactericidal domains in the full-length molecule or promote liberationof bactericidal fragments the full-length molecule by a mechanism suchas bacterial protease action or by autoproteolysis, but the inventors donot wish to be bound by this theory. The results disclosed hereinsupport the notion that bactericidal serine esterases possess broadspectrum antibacterial action due to the presence of internalantibacterial domains and that multiple, distinct domains exist withincathepsin G for the purpose of killing different pathogens.

The following examples are provided for illustrative purposes and arenot intended to limit the scope of the invention. Because modificationof the examples below will be apparent to those of skill in the art, itis intended that this invention be limited only by the scope of theappended claims. All references cited in this application are herebyincorporated by reference herein.

EXAMPLES Example 1.1

Purification of Cat G and Human Neutrophil Elastase

Cat G and human neutrophil elastase (HLE) were purified from extracts ofhuman PMN granules as described previously (Baugh and Travis (1976)Biochemistry 15:836-841). After purification, each enzyme was stored at-20° C. in 50 mM sodium acetate (pH 5.5) 0.5M NaCl prior to use.

Example 1.2

Clostripain Digestion of Cathepsin G

Cat G (600 micrograms) was incubated with purified clostripain (SigmaChemical Company, St. Louis, Mo.) at a molar ratio of 50:1 in 50 mMTris-HCl (pH 7.5) 10 mM CaCl₂ 0.16M NaCl 2.5 mM Dithiothreitol at 37° C.for 24 hours. During the incubation period samples were removed atvarious times and assayed for enzymatic and/or bacterial activity.

The enzymatic digestion of Cat G was monitored by loss of esteraseactivity using the synthetic substrate (Suc-L-Ala-L-Ala-Pro-Phe-pNA(Nakajima et al. (1979) J. Biol. Chem. 254:4027-4032). In some instancesdigestion was monitored by SDS polyacrylamide gel electrophoresis(Laemmli (1970) Nature (London) 227:680-685). Cat G was tested forantimicrobial activity as described in Shafer et al. (1986) Infect.Immun. 54:184-188.

Example 1.3

Isolation Of Cat G Peptides from Clostripain Digest

Clostripain (Mr 30 kDa) and undigested Cat G were separated from thedegradation peptides by loading the 24 h digestion mixture (1.0 ml) on aSephadex G-50™ (Pharmacia Fine Chemicals, Piscataway, N.J.) column whichwas equilibrated with 1.0M NH₄₀ H. Absorbances at 220 nm and 280 nm weremonitored and those fractions containing peptides were pooled,lyophilized and dissolved in 0.1% trifluoroacetic acid (TFA).

The peptide mixture was then applied to a reverse phase HPLC (RP-HPLC)C-18-10 column which had been previously equilibrated with 0.1% TFA.Bound peptides were then eluted using a linear gradient of acetonitrile(0 to 70% (v/v)) in 0.1% TFA at a flow rate of 0.5 ml/min. Fractionswere lyophilized for subsequent testing in antimicrobial assays.Fractions with bactericidal activity were reapplied to the RP-HPLCC-18-10 column. The bound peptides were eluted with non-linear gradientof acetonitrile (0 to 50% v/v) in 0.1% TFA with a flow rate of 0.5 ml/h.The elution of peptides was monitored by following A₂₂₀. Peptideconcentrations were measured using ninhydrin as described (Rosen et al.(1962) Anal. Biochem. 4:213-221).

Example 1.4

Antimicrobial Activity Testing

Neisseria gonorrhoeae strain FA 102 and Staphylococcus aureus strain8325-4 were the test bacteria used in many experiments; these strainshave been described previously (Shafer et al. (1986) supra; Shafer andOnunka (1989) J. Gen. Microbiol. 135:825-830). N. gonorrhoeae werepassaged on clear typing agar as nonpiliated, transparent variants. Fortesting, cultures were grown with shaking at 37° C. in GC brothcontaining glucose, iron and sodium bicarbonate supplements. S. aureuswas grown at 37° C. with shaking in LB broth. At midlogarithmic phase(OD₅₅₀ of 0.35) the cultures were diluted in Hanks Balanced SaltSolution (HBSS) (Gibco Laboratories, Grand Island, N.Y.) (pH 7.5) togive approximately 10⁵ CFU/ml. In other experiments, P. aeruginosa ATCC27853, a standard antibiotic tester strain, was used.

Peptides were dissolved in HBSS (pH 7.5) and added in various amounts (0to 100 micrograms) to sterile microtiter wells. After UV sterilizationof the wells, 0.1 ml samples of the bacterial were added and the volumesin each well were adjusted with HBSS to 0.2 ml. The bacteria-peptidemixtures were incubated at 37° C. for 45-60 min unless otherwise noted.For N. gonorrhoeae, incubation was carried out under an atmosphere of 5%CO₂. In other experiments, as noted, 1/100 strength HBSS was used. Forat least some strains, the use of 1/100 HBSS resulted in greatersensitivity to the bactericidal activity of the peptides disclosedherein.

Viability was determined after incubation by plating 10 and 100microliter samples on LB agar (S. aureus) or GCB agar (N. gonorrhoeae).All assays were done in duplicate or triplicate, and the results givenare the means of three independent experiments. The % survival of thetest bacteria was calculated as 100×(# CFU in the presence ofpeptide)/(# CFU in the absence of peptide); standard error of the meanfor each data point was never greater than 5%.

Certain antibiotics were tested for antibacterial activity using theprocedure described above. Penicillin G, tetracycline, chloraphenicol,streptomycin and kanamycin (Sigma Chemical Company, St. Louis, Mo.) wereeach dissolved in distilled water or 50% (v/v) methanol to give a 1mg/ml solution. All antibiotic solutions were stored at -20° C.

A partially purified preparation containing the three human defensinsdescribed by Selsted et al. (1985) J. Clin. Invest. 76:1436-1439, wasalso used in the antibacterial activity testing protocol describedabove. This preparation was provided by Dr. John Spitznagel, EmoryUniversity School of Medicine, Atlanta, Ga.). The preparation wasobtained by Sephadex G-75™ (Pharmacia, Piscataway, N.J.) chromatographyof a crude acid extract of human PMN granules (Shafer et al. (1988)Infect. Immun. 56:51-53). The mixture of the three defensins eluted fromthe column after lysozyme (Mr 14.4 kDa), as is consistent with theirmolecular weights of approximately 5 kDa. Before use in theantibacterial activity test, the pooled defensins were dialyzed against4 liters of distilled water at 4° C. using dialysis tubing with anexclusion limit of 3 kDa.

Example 1.5

Sequence analysis of Antibacterial Peptides

The sequences of the antibacterial peptides from clostripain digestionand HPLC resolution were determined by automated Edman degradation in aModel 477A Pulse Liquid Sequencer (Applied Biosystems, Inc., FosterCity, Calif.), and the PTH-amino acids were identified on-line in a 120APTH Analyzer (Applied Biosystems, Inc.).

Example 2

Preparation of Synthetic peptides

Oligopeptides were synthesized using an Applied Biosystems Model 430Apeptide synthesizer (0.1-0.5 mmol scale) using phenylacetamidomethyl(Pam) or p-methylbenzyhydrylamine copoly(styrene/divinylbenzene) resins(Applied Biosystems, Inc., Foster City, Calif.) andtert-butyloxycarbonyl (Boc)-protected amino acids (Applied Biosystems,Inc. or Bachem, Inc., Torrance, Calif.). Boc-N-methyl-Ala, Boc-Arg(tosyl) or Boc-Arg (mesitylenesulfonyl), Boc-Asp (benzyl), Boc-Cys(4-methoxybenzyl), Boc-Glu (benzyl), Boc-His (benzyloxycarbonyl) orBoc-His (2,4-dinitrophenyl ), Boc-DHis(4-toluenesulfonyl),Boc-Lys(chlorobenzyloxycarbonyl), Boc-Met, Boc-Ser (benzyl), Boc-Thr(benzyl), Boc-Trp or Boc-Trp (formyl), and Boc-Tyr(2-bromobenzyloxycarbonyl) were used for the incorporation of therespective amino acid residues. Boc-His(methyl) was incorporated in amanual mode on a 0.02 mmol scale using theN,N-dicyclohexylcarbodiimide/1-hydroxybenzotriazole coupling protocol.All amino acids (except glycine) used herein have the L configurationunless otherwise noted.

Peptides were cleaved from the resin and deprotected in liquidHF/p-cresol/dimethyl sulfide (10:1:0.5) at -5° C. for 90 min, or inliquid HF/anisole (9:1, v/v) at 0° C. for 90 min. The resins were washedwith cold diethyl ether, and the peptides were extracted into 1.0Macetic acid and lyophilized. Peptides were then purified by RP-HPLC onan Aquapore™ RP-300 C18 silica column (1×10 cm, Applied Biosystems,Inc.), or on an MRPH-Gel™ polystyrene column (1×10 cm, The Nest Group,Scarborough, Mass.) using a 0-60% linear gradient of acetonitrile in0.1% TFA. The purity of each synthetic peptide preparation was confirmedby microbore HPLC on Aquapore™ OD-300 columns of C18 silica (1×250 mm,Applied Biosystems, Inc.), quantitative amino acid analysis andsequencing, as described above. Peptides were generally stored in thelyophilized form at 4° C. prior to use in the antibacterial assays.

It is understood in the art that there are other suitable peptidesynthetic devices or that manual peptide synthesis could be carried outto produce the peptides of the present invention. Automated solid phasepeptide synthesis is described, e.g., in Stewart et al. (1984) SolidPhase peptide Synthesis, Pierce Chemical Company, Rockford, Ill.).

Example 3

Search for Related Peptide Sequences

A search was done on protein computer databases using the HPQYNQRsequence for similar sequences in other proteins. Sequences with somesimilarity to this Cat G sequence were recognized in certain othercytotoxic proteases. Searches were also done using the IIGGR sequence(SEQ ID NO:1) and the CG 117-136 sequence (SEQ ID NO:22).

The antimicrobial peptides of the present invention will be useful forinhibiting bacterial growth for research, including in vitro cultureapplications, and, when formulated into therapeutic compositions, willbe useful in the treatment of infections, especially bacterialinfections. The antimicrobial peptides can be administered by anymechanism known to the art, as appropriate for a particular type ofinfection.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 59                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       Ile IleGlyGlyArg                                                              15                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       IleValGlyGlyArg                                                                15                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       HisProGlnTyrAsnGlnArg                                                         1 5                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 2..3                                                            (D) OTHER INFORMATION: /label=Xaa                                             /note="X at position 2 is Pro, His or Ala."                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..3                                                            (D) OTHER INFORMATION: /product="OTHER"                                       /label=Xaa                                                                    /note="X at position 3 is Asp, Asn, Glu, Gln, Ala, Ser,                       Thr, Ile, Val, Tyr, Arg, Methionin Oxide or Methionine                        Sulfone."                                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                           (B) LOCATION: 3..4                                                           (D) OTHER INFORMATION: /product="OTHER"                                       /label=Xaa                                                                    /note="X at position 4is Tyr, Phe, Trp or                                     Beta- naphthyl-alanine."                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 4..5                                                            (D) OTHER INFORMATION: /product="OTHER"                                       /label=Xaa                                                                     /note="X at position 5 is Ala or Asn."                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 5..6                                                            (D) OTHER INFORMATION: /product="OTHER"                                       /label=Xaa                                                                    /note="X at position 6 is Gln, Pro, Ala or                                    N-methyl- alanine."                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B ) LOCATION: 6..7                                                           (D) OTHER INFORMATION: /product="OTHER"                                       /label=Xaa                                                                    /note="X at position 7 is Arg, Lys, Ala, NH2 or OH."                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       HisXaaXaaXaaXaaXaaXaa                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                      (B) TYPE: amino acid                                                         (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       HisProAlaTyrAsnProLys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       HisProAlaTyrAsnProArg                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       HisProAlaTyrAsnGlnArg                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       HisProGlnTyrAlaGlnArg                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       HisProGlnTyrAsnGlnAla                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      HisProGlnTyrAsnAlaArg                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      HisA laGlnTyrAsnGlnArg                                                        15                                                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      HisProGlnTyrAsn Gln                                                           15                                                                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      ArgHisProGlnTyrAsnGlnArg                                                      1 5                                                                           (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      HisProGlnTyrAsnGlnArgThrIleGlnAsnAspIl eMetLeuLeu                             151015                                                                        GlnLeuSerArg                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AlaProGlnTyrAsnGlnArg                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D ) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      IleIleGlyGlyArgGluSerArgProHisSerArgProTyrMetAla                              151015                                                                        TyrLeuGlnIle                                                                   20                                                                           (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GlnSerProAlaGlyGlnSerArgCysGlyGlyPheLeuV alArgGlu                             151015                                                                        AspPheValLeu                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      ThrAlaAlaHisCysTrpGlySerAsnIleAsnValThrLeuGlyAla                              151015                                                                        HisAsnIle Gln                                                                 20                                                                            (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      ArgArgGluAsnThrGlnGlnHisIleThrA laArgArgAlaIleArg                             151015                                                                        HisProGlnTyr                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      HisProGlnTyrAsnGlnArgThrIleGlnAsnAspIleMetLeuLeu                              151015                                                                         GlnLeuSerArg                                                                 20                                                                            (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      ArgValArgArgAsnArgAsnV alAsnProValAlaLeuProArgAla                             151015                                                                        GlnGluGlyLeu                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    ( B) TYPE: amino acid                                                         (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      ArgProGlyThrLeuCysThrValAlaGlyTrpGlyArgValSerMet                              1510 15                                                                       ArgArgGlyThr                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      AspThrLeuArgG luValGlnLeuArgValGlnArgAspArgGlnCys                             151015                                                                        LeuArgIlePhe                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                     (B) TYPE: amino acid                                                         (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      GlySerTyrAspProArgArgGlnIleCysValGlyAspArgArgGlu                              1510 15                                                                       ArgLysAlaAla                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      PheL ysGlyAspSerGlyGlyProLeuLeuCysAsnAsnValAlaHis                             151015                                                                        GlyIleValSerTyr                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 27 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      GlyLysSerSerGlyValProProGluValPheThrArgPheValSer                              15 1015                                                                       SerPheLeuProTrpIleArgThrThrMetArg                                             2025                                                                          (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          ( C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      AlaProGlnTyrAsnGlnArg                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      IleIleGlyGlyHis                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                     IleIleGlyGlyVal                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      Hi sProGlnTyrAsnProGln                                                        15                                                                            (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      HisHisGlnTyrA snGlnArg                                                        15                                                                            (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      HisProGlnAlaAsnGlnArg                                                          15                                                                           (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      HisProGlnLysAsnThrTyr                                                         1 5                                                                           (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      HisProGlnPheAsnGlnArg                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      HisProAsnTyrAsnGlnArg                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 7 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      HisProGluTyrAsnGlnArg                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      HisProLeuTyrAsnGlnArg                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                     (B) TYPE: amino acid                                                         (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      ArgProGlyLeuThrLeuCysThrValAlaGlyTrpGly                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 15 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      CysThrValAlaGlyTrpGlyArgValSerMetArgArgGlyThr                                 15 1015                                                                       (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      TrpGlyArgValSerMetArgArgGl yThr                                               1510                                                                          (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 223 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      IleIleGlyGly ArgGluSerArgProHisSerArgProTyrMetAla                             151015                                                                        TyrLeuGlnIleGlnSerProAlaGlyGlnSerArgCysGlyGlyPhe                               202530                                                                       LeuValArgGluAspPheValLeuThrAlaAlaHisCysTrpGlySer                              354045                                                                        AsnIleAsnValThr LeuGlyAlaHisAsnIleAspArgArgGluAsn                             505560                                                                        ThrGlnGlnHisIleThrAlaArgArgAlaIleArgHisProGlnTyr                              6570 7580                                                                     AsnGlnArgThrIleGlnAsnAspIleMetLeuLeuGlnLeuSerArg                              859095                                                                        ArgValArgArgAsn ArgAsnValAsnProValAlaLeuProArgAla                             100105110                                                                     GlnGluGlyLeuArgProGlyThrLeuCysThrValAlaGlyTrpGly                              115 120125                                                                    ArgValSerMetArgArgGlyThrAspThrLeuArgGluValGlnLeu                              130135140                                                                     ArgValGlnArgAspArgGlnCysL euArgIlePheGlySerTyrAsp                             145150155160                                                                  ProArgArgGlnIleCysValGlyAspArgArgGluArgLysAlaAla                              165 170175                                                                    PheLysGlyAspSerGlyGlyProLeuLeuCysAsnAsnValAlaHis                              180185190                                                                     GlyIleValSerTyrGl yLysSerSerGlyValProProGluValPhe                             195200205                                                                     ThrArgValSerSerPheLeuProTrpIleArgThrThrMetArg                                 210215 220                                                                    (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      LysProGlyGlnThrCysSerValAlaGlyTrpGl yGlnThrAlaPro                             151015                                                                        LeuGlyLysSer                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      LysProGlnAspValCysTyrValAlaGlyTrpGlyArgMetAlaPro                              151015                                                                        Met GlyLysTyr                                                                 20                                                                            (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      GluAlaGlnThrArgCysGlnValAl aGlyTrpGlySerGlnSerArg                             151015                                                                        SerGlyGlyArg                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      GlyAsnGlyValGlnCysLeuAlaMetGlyTrpGlyLeuLeuGlyArg                              151015                                                                        AsnArgGlyIle                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      ProHisGlyThrGlnCy sLeuAlaMetGlyTrpGlyArgValGlyAla                             151015                                                                        HisProProPro                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                     (B) TYPE: amino acid                                                         (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      AlaAlaGlyThrThrCysValThrThrGlyTrpGlyLeuThrArgTyr                              1510 15                                                                       ThrAsnAlaAsn                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      GlyIleGl yLysPheLeuHisSerAlaLysLysPheLysAlaPheVal                             151015                                                                        GlyGluIleMetAsn                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 7 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      HisProGlnTyrAsnProLys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                      (B) TYPE: amino acid                                                         (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      HisProGlnTyrAsnProArg                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      TyrProCysTyrAspProAla                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      HisProAlaTyrAsnAlaLys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      HisProAspTyrAsnGlnArg                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      TyrProCysTyrAspGluTyr                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      HisProAspTyrAsnProLys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      HisP roAspTyrAsnProAsp                                                        15                                                                            (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      HisProAspTyrAsn AlaThr                                                        15                                                                            (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      HisProAlaTyrAspAspLys                                                         1 5                                                                           (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      HisProAlaPheAspArgLys                                                         1 5                                                                       

We claim:
 1. a peptide having antimicrobial activity, said peptidehaving a length of from five amino acids up to twenty-six amino acids,and which peptide comprises a sequence selected from the group of aminoacid sequences consisting of IIGGR (SEQ ID NO:1), IVGGR (SEQ ID NO:2)and HPQYNQR (SEQ ID NO:3).
 2. A therapeutic composition for controllinginfection by a bacterium, said composition comprising at least oneantimicrobial peptide of claim 1 and a pharmacologically acceptablecarrier, wherein said bacterium is sensitive to the antimicrobialactivity of said peptide.
 3. A peptide having antimicrobial activity,said peptide having from ten to twenty-seven amino acids, and saidpeptide having an amino acid sequence selected from the group of aminoacid sequences consisting of IIGGRESRPHSRPYMAYLQI (SEQ ID NO:16),RRENTQQHITARRAIRHPQY (SEQ ID NO:19), RPGTLCTVAGWGRVSMRRGT (SEQ IDNO:19), GKSSGVPPEVFTRFVSSFLPWIRTTMR (SEQ ID NO:26), CTVAGWGRVSMRRGT (SEQID NO:39), WSRVSMRRGT (SEQ ID NO:40) and RPGTLCTVAGWGRVSMRRGT, whereineach amino acid of the latter is in the D-configuration.
 4. Atherapeutic composition for controlling infection by a bacterium, saidcomposition comprising at least one antimicrobial peptide of claim 3 anda pharmacologically acceptable carrier, wherein said bacterium issensitive to the antimicrobial activity of said peptide.